Project Summary: Oropharyngeal and esophageal candidiasis are mainly caused by the opportunistic pathogen Candida albicans and continue to be a health issue in immunocompromised populations. Recurrent infections of C. albicans are often resistant to multiple major antifungal drug types. In these cases there are few alternative therapies since clinically available antifungals target a limited number of fungal pathways. Salivary Histatin 5 (Hst 5) is secreted by human major salivary glands and has high in vitro fungicidal activity against C. albicans and other Candida species. Hst 5 has a mechanism of action that is distinct from antifungal drugs currently in use, making it an attractive alternative treatment; however the ultimate mechanism of killing is unclear. Hst 5 treatment induces several effects on C. albicans cells, including reactive oxygen species (ROS) accumulation, activation of the high osmolarity glycerol (HOG1) pathway and cell cycle arrest in G1, however its underlying mechanism of action is still mostly unknown. Importantly, Hst 5 is able to bind zinc (Zn2+) and copper (Cu2+) ions with high affinity and binding of these metals has been shown to impact killing activity for unknown reasons. Entry of Hst 5 into fungal cells is required for its high killing activity, and free metal binding sites appear to be necessary for uptake of the peptide; therefore the central hypothesis of this project is that Hst 5 enters fungal cells to deplete intracellular Zn2+ and Cu2+, which is the mechanism by which Hst 5 induces oxidative stress, changes in HOG1 signaling, and G1 cell cycle arrest in C. albicans. As a tool to probe the metal-dependent effects of Hst 5, a mutant Hst 5 peptide that is designed to be deficient in metal binding will be used. In Aim 1, the ability of Hst 5 to deplete intracellular Zn2+ and Cu2+ will be verified, and the expression and activity of ROS-detoxifying superoxide dismutase proteins during Hst 5 mediated metal depletion will be characterized. In Aim 2 the ability of Zn2+ to regulate HOG1 pathway signaling and cell cycle arrest will be tested, and it will be confirmed that Hst 5 treatment dysregulates these processes through intracellular Zn2+ depletion. Completion of this project will be a valuable step forward in research into the Hst 5 mechanism of action, and provide new characterization of C. albicans stress responses.